Synthetic methylotrophs and uses thereof

Abstract

The present invention provides a method for increasing production of a metabolite by a non-naturally occurring methylotroph, comprising growing the non-naturally occurring methylotroph in a medium comprising methanol. Expression of one or more native genes in the non-naturally occurring methylotroph is changed. Also provided are the non-naturally occurring methylotroph and preparation thereof.

1 . A method for increasing production of a metabolite by a non-naturally occurring methylotroph, comprising growing the non-naturally occurring methylotroph in a medium comprising methanol, wherein expression of one or more native genes in the non-naturally occurring methylotroph is changed. 
     
     
         2 . The method of  claim 1 , wherein the one or more native genes comprise 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase gene (gpmA), 2,3-bisphosphoglycerate-independent phosphoglycerate mutase gene (gpmM), 2-dehydro-3-deoxy-D-gluconate 6-phosphate (KDPG) aldolase gene (eda), 6-phosphogluconate dehydrogenase gene (gnd), aminomethyltransferase gene (gcvT), bifunctional (p)ppGpp synthetase II/guanosine-3′,5′-bis pyrophosphate 3′-pyrophosphohydrolase gene (spoT), enolase gene (eno), fructose-1,6-bisphosphatase 1 class 2 gene (glpX), fructose-1,6-bisphosphatase class 1 gene (fbp), GDP pyrophosphokinase/GTP pyrophosphokinase gene (relA), glucose-6-phosphate isomerase gene (pgi), glyceraldehyde-3-phosphate dehydrogenase gene (gapA), glyceraldehyde-3-phosphate dehydrogenase gene (gapC), glycine cleavage system H protein gene (gcvH), glycine decarboxylase gene (gcvP), HTH-type transcriptional regulator GntR gene (gntR), leucine-responsive regulatory protein gene (lrp), methylglyoxal synthase gene (mgsA), phosphogluconate dehydratase gene (edd), phosphoglycerate dehydrogenase gene (serA), phosphoglycerate kinase gene (pgk), ribulose-phosphate 3-epimerase gene (rpe), RNA polymerase-binding transcription factor gene (dksA), serine hydroxymethyltransferase gene (glyA), transaldolase A gene (talA), transaldolase B gene (talB), or a combination thereof. 
     
     
         3 . The method of  claim 1 , wherein the one or more native genes are deleted from the genome of the non-naturally occurring methylotroph, wherein the one or more native genes comprise leucine-responsive regulatory protein gene (lrp). 
     
     
         4 . The method of  claim 1 , wherein the one or more native genes comprise one or more deletions. 
     
     
         5 . The method of  claim 1 , wherein the medium further comprises a co-substrate. 
     
     
         6 . The method of  claim 5 , wherein the co-substrate comprises one or more monosaccharides selected from the group consisting of glucose, xylose, mannose, arabinose, rhamnose, ribose and a combination thereof. 
     
     
         7 . The method of  claim 5 , wherein the co-substrate comprises one or more amino acids selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine and a combination thereof. 
     
     
         8 . The method of  claim 1 , wherein the non-naturally occurring methylotroph is a microbe derived from a microbe selected from the group consisting of  Acetobacter, Acinetobacter, Bacillus, Chlorobi, Clostridium, Corynebacterium, Cyanobacteria, Deinococcus, Enterobacter, Enterobacteria, Escherichia, Geobacillus, Geobacter, Klebsiella, Lactobacillus, Lactococcus, Mannheimia, Propionibacterium, Pseudomonas, Ralstonia, Shewanella, Streptococcus, Streptomyces, Synechococcus, Synechocystis  and  Zymomonas.    
     
     
         9 . The method of  claim 1 , wherein the non-naturally occurring methylotroph is  Escherichia coli.    
     
     
         10 . The method of  claim 1 , further comprising incorporating a carbon atom from the methanol into the metabolite. 
     
     
         11 . The method of  claim 1 , wherein the metabolite is selected from the group consisting of 4-carbon chemicals, diacids, 3-carbon chemicals, higher carboxylic acids [which may include acetic acid, propionic acid, butyric acid, valeric (pentanoic) acid and caproic (hexanoic) acid], alcohols of higher carboxylic acids and polyhydroxyalkanoates. 
     
     
         12 . The method of  claim 1 , wherein the metabolite is selected from the group consisting of acetone, isopropanol, 1,3-butanediol and n-butanol. 
     
     
         13 . The method of  claim 1 , wherein the metabolite is acetone, further comprising expressing a heterologous enzyme having an acetyl-CoA C-acetyltransferase activity [e.g., thiolase (EC 2.3.1.9) (THL)], a heterologous enzyme having a butyrate-acetoacetate CoA-transferase activity [e.g., acyl(acetate/butyrate)-acetoacetate coenzyme A transferase (EC 2.8.3.9) (CTFA/B), and a heterologous enzyme having acetoacetate decarboxylase activity [e.g., acetoacetate decarboxylase (EC 4.1.1.4) (ADC) in the non-naturally occurring methylotroph. 
     
     
         14 . The method of  claim 1 , further comprising increasing the production of the metabolite by the non-naturally occurring methylotroph by at least 5% as compared with that by a control microbe in which the expression of the one or more native genes is not changed. 
     
     
         15 . A non-naturally occurring methylotroph, wherein expression of one or more native genes in the non-naturally occurring methylotroph is changed, wherein the one or more native genes comprise 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase gene (gpmA), 2,3-bisphosphoglycerate-independent phosphoglycerate mutase gene (gpmM), 2-dehydro-3-deoxy-D-gluconate 6-phosphate (KDPG) aldolase gene (eda), 6-phosphogluconate dehydrogenase gene (gnd), aminomethyltransferase gene (gcvT), bifunctional (p)ppGpp synthetase II/guanosine-3′,5′-bis pyrophosphate 3′-pyrophosphohydrolase gene (spoT), enolase gene (eno), fructose-1,6-bisphosphatase 1 class 2 gene (glpX), fructose-1,6-bisphosphatase class 1 gene (fbp), GDP pyrophosphokinase/GTP pyrophosphokinase gene (relA), glucose-6-phosphate isomerase gene (pgi), glyceraldehyde-3-phosphate dehydrogenase gene (gapA), glyceraldehyde-3-phosphate dehydrogenase gene (gapC), glycine cleavage system H protein gene (gcvH), glycine decarboxylase gene (gcvP), HTH-type transcriptional regulator GntR gene (gntR), leucine-responsive regulatory protein gene (lrp), methylglyoxal synthase gene (mgsA), phosphogluconate dehydratase gene (edd), phosphoglycerate dehydrogenase gene (serA), phosphoglycerate kinase gene (pgk), ribulose-phosphate 3-epimerase gene (rpe), RNA polymerase-binding transcription factor gene (dksA), serine hydroxymethyltransferase gene (glyA), transaldolase A gene (talA), transaldolase B gene (talB), or a combination thereof.